My doctoral research primarily focuses on the Escherichia coli hotdog-fold thioesterases ydiI and ybgC.
The ydiI gene is colocalized in the operon ydiHIJ. A substrate screen provided evidence that YdiI prefers aryl-CoA substrates and discriminated against the analogous aryl-ACP, unlike the paralog YbdB which works on 2,4-DHB-EntB in the enterobactin synthesis pathway. A bioinformatic approach showed that in some bacteria, ydiI is colocalized with genes from the menaquinone pathway. YdiI was shown to catalyze the hydrolysis of DHNA-CoA with physiological relevance (kcat/KM ~ 105). Furthermore, the E. coli YdiI- strain was shown to have perturbed growth, in good agreement with other menaquinone enzyme knockout experiments. Taking into account the evidence provided, YdiI is likely the DHNA-CoA thioesterase in the menaquinone pathway.
Within the substrate binding pocket, changes to the YdiI Val68 to the YbdB equivalent of Met resulted in perturbed catalytic efficiency towards lauroyl-CoA, however not towards its physiological substrate DHNA-CoA. The YbdB M68V mutant resulted in increased efficiency towards lauroyl-CoA and DHNA-CoA, seemingly making it a gatekeeper. Secondly, YdiI catalyzes the hydrolysis of acyl-CoA's using a Glu63, His54, and Gln48 triad determined by mutagenesis experiments. The utilization of 18-O incorporation and rapid-quench techniques provided insight into YdiI's catalytic mechanism. A single phase multiple turnover reaction and the incorporation of 18-O in single turnover reactions suggests that ydiI uses a general base catalytic mechanism where Glu63 is the activating residue.
The second E. coli hotdog-fold thioesterase protein discussed within is YbgC. YbgC is encoded by the first ORF of the tol-pal gene cluster. Previous work on the H. influenzae homologue demonstrated affinity for short chain acyl-CoA's and tandem affintity purificiation experiments showed E. coli YbgC co-purifies with ACP. No previous attempts to screen for acyl-ACP have been made. To screen YbgC for ACP activity, a method was developed to stoichiometrically construct acyl-ACP's, using apoACP, BF1558 acyl-transferase and acyl-CoA. The E. coli YbgC demonstrated preference for long chain acyl-CoA's and their analogous acyl-ACPs. To understand the nature of ACP binding to YbgC, SAXS analysis was carried out on the complex demonstrating that 2 ACP molecules bind to 1 dimer of YbgC.